Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 21, 2015; 21(7): 2011-2029
Published online Feb 21, 2015. doi: 10.3748/wjg.v21.i7.2011
Gene expression profiling of MYC-driven tumor signatures in porcine liver stem cells by transcriptome sequencing
Rajagopal N Aravalli, Neil C Talbot, Clifford J Steer
Rajagopal N Aravalli, Department of Radiology, University of Minnesota Medical School, Minneapolis, MN 55455, United States
Neil C Talbot, Beltsville Agricultural Research Center, US Department of Agriculture, Beltsville, MD 20705, United States
Clifford J Steer, Departments of Medicine, and Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 54455, United States
Author contributions: Aravalli RN designed the study, performed all the experiments, and wrote the manuscript; Talbot NC provided PICM-19 cells; Steer CJ provided vital resources to carry out the proposed studies; all authors reviewed and revised the manuscript.
Supported by Departmental funds to Dr. Aravalli RN
Ethics approval: Institutional review board approval is not needed for this study since it does not involve any human subjects. Therefore, we do not have any documents to enclose with this revision.
Institutional animal care and use committee: All procedures involving mice were reviewed and approved by the institutional animal care and use Committee of the University of Minnesota (IACUC protocol number: 1107A01962).
Conflict-of-interest: All authors have no conflict of interest. We have included the funding source in the title page of the revised manuscript.
Data sharing: Additional information of RNA-seq dataset is available from the corresponding author at arava001@umn.edu.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Rajagopal N Aravalli, PhD, Department of Radiology, University of Minnesota Medical School, MMC 292 Mayo, 420 Delaware Street SE, Minneapolis, MN 55455, United States. arava001@umn.edu
Telephone: +1-61-26265540 Fax: +1-61-26265580
Received: September 4, 2014
Peer-review started: September 4, 2014
First decision: October 29, 2014
Revised: November 6, 2014
Accepted: December 14, 2014
Article in press: December 16, 2014
Published online: February 21, 2015
Processing time: 160 Days and 9.9 Hours
Abstract

AIM: To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells.

METHODS: In this study, we have used an immortal porcine liver stem cell line, PICM-19, to study the role of c-MYC in hepatocarcinogenesis. PICM-19 cells were converted into cancer cells (PICM-19-CSCs) by overexpressing human MYC. To identify MYC-driven differential gene expression, transcriptome sequencing was carried out by RNA sequencing, and genes identified by this method were validated using real-time PCR. In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice.

RESULTS: Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells (PICM-19-CSCs). By using comparative bioinformatics analyses, we have determined that > 1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs. Gene ontology analysis further showed that the MYC-induced, altered gene expression was primarily associated with various cellular processes, such as metabolism, cell adhesion, growth and proliferation, cell cycle, inflammation and tumorigenesis. Interestingly, six genes expressed by PICM-19 cells (CDO1, C22orf39, DKK2, ENPEP, GPX6, SRPX2) were completely silenced after MYC-induction in PICM-19-CSCs, suggesting that the absence of these genes may be critical for inducing tumorigenesis.

CONCLUSION: MYC-driven genes may serve as promising candidates for the development of hepatocellular carcinoma therapeutics that would not have deleterious effects on other cell types in the liver.

Keywords: Hepatocellular carcinoma; MYC; Stem cells; Gene expression; RNA sequencing

Core tip: It is well-established that cancer stem cells not only drive tumor growth in the liver, in general, but also that the proto-oncogene c-MYC plays a critical role in that process. However, little is known about genes induced and regulated by MYC to generate tumors, and, in particular, those involved in liver stem cells. In this study, we examined the role of MYC protein in hepatocarcinogenesis using an immortal porcine liver stem cell line, PICM-19. Interestingly, MYC-overexpression silenced the expression of six genes in PICM-19 cells (CDO1, C22orf39, DKK2, ENPEP, GPX6, SRPX2), suggesting that they may be critical in inducing tumorigenesis.